Alk1 receptor and ligand antagonists and uses thereof

ABSTRACT

In certain aspects, the present disclosure relates to the insight that a polypeptide comprising a ligand-binding portion of the extracellular domain of activin-like kinase I (ALK1) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders. The disclosure also identifies ligands for ALK1 and demonstrates that such ligands have pro-angiogenic activity, and antibodies that inhibit receptor-ligand interaction.

RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.11/982,738, filed on Nov. 2, 2007, which claims the benefit of thefiling date under 35 U.S.C. §119(e) of U.S. provisional application60/856,592, filed Nov. 2, 2006, the entire contents of each of which areincorporated herein by reference.

BACKGROUND

Angiogenesis, the process of forming new blood vessels, is critical inmany normal and abnormal physiological states. Under normalphysiological conditions, humans and animals undergo angiogenesis inspecific and restricted situations. For example, angiogenesis isnormally observed in wound healing, fetal and embryonic development andformation of the corpus luteum, endometrium and placenta.

Undesirable or inappropriately regulated angiogenesis occurs in manydisorders, in which abnormal endothelial growth may cause or participatein the pathological process. For example, angiogenesis participates inthe growth of many tumors. Deregulated angiogenesis has been implicatedin pathological processes such as rheumatoid arthritis, retinopathies,hemangiomas, and psoriasis. The diverse pathological disease states inwhich unregulated angiogenesis is present have been categorized asangiogenesis-associated diseases.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Capillary blood vessels are composed primarily ofendothelial cells and pericytes, surrounded by a basement membrane.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. The endothelial cells,which line the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic factors induce the endothelial cells tomigrate through the eroded basement membrane. The migrating cells form a“sprout” protruding from the parent blood vessel, where the endothelialcells undergo mitosis and proliferate. Endothelial sprouts merge witheach other to form capillary loops, creating the new blood vessel.

Agents that inhibit angiogenesis have proven to be effective in treatinga variety of disorders. Avastin™ (bevacizumab), a monoclonal antibodythat binds to Vascular Endothelial Growth Factor (VEGF), has proven tobe effective in the treatment of a variety of cancers. Macugen™, anaptamer that binds to VEGF has proven to be effective in the treatmentof neovascular (wet) age-related macular degeneration. Antagonists ofthe SDF/CXCR4 signaling pathway inhibit tumor neovascularization and areeffective against cancer in mouse models (Guleng et al. Cancer Res. 2005Jul. 1; 65(13):5864-71). The isocoumarin2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3)has completed phase I clinical evaluation as an orally bioavailableangiogenesis inhibitor. NM-3 directly kills both endothelial and tumorcells in vitro and is effective in the treatment of diverse human tumorxenografts in mice (Agata et al. Cancer Chemother Pharmacol. 2005December; 56(6):610-4.). Thalidomide and related compounds have shownbeneficial effects in the treatment of cancer, and although themolecular mechanism of action is not clear, the inhibition ofangiogenesis appears to be an important component of the anti-tumoreffect (see, e.g., Dredge et al. Microvasc Res. 2005 January;69(1-2):56-63). The success of TNF-alpha antagonists in the treatment ofrheumatoid arthritis is partially attributed to anti-angiogenic effectson the inflamed joint tissue (Feldmann et al. Annu Rev Immunol. 2001;19:163-96). Anti-angiogenic therapies are widely expected to havebeneficial effects on other inflammatory diseases, particularlypsoriasis. Although many anti-angiogenic agents have an effect onangiogenesis regardless of the tissue that is affected, other angiogenicagents may tend to have a tissue-selective effect.

It is desirable to have additional compositions and methods forinhibiting angiogenesis. These include methods and compositions whichcan inhibit the unwanted growth of blood vessels, either generally or incertain tissues and/or disease states.

SUMMARY

In part, the present disclosure presents a characterization of anactivin-like kinase I (ALK1)-mediated regulatory system and the role ofthis system in angiogenesis in vivo. In certain aspects, the disclosureprovides antagonists of ALK-1 ligands and the use of such antagonists asanti-angiogenic agents. Additionally, the disclosure providesantagonists of ALK-1 itself, and the use of such antagonists asanti-angiogenic agents. As described herein, ALK1 is a receptor for theGDF5 group of ligands, which includes GDF6 and GDF7, and also for theBMP9 group of ligands, which includes BMP10. This disclosuredemonstrates that signaling mediated by ALK1 and the ligands describedabove is involved in angiogenesis in vivo, and that inhibition of thisregulatory system has a potent anti-angiogenic effect. Thus, in certainaspects, the disclosure provides antagonists of the ALK1 regulatorysystem, including antagonists of the receptor or one or more of theligands, for use in inhibiting angiogenesis. In certain aspects, thedisclosure provides antagonists of ALK1 ligands for the treatment ofcancers, particularly multiple myeloma, rheumatoid arthritis, anddisorders associated with pathological angiogenesis in the eye.

In certain aspects, the disclosure provides polypeptides comprising aligand binding portion of the extracellular domain of ALK1 (“ALK1 ECDpolypeptides”) for use in inhibiting angiogenesis. While not wishing tobe bound to any particular mechanism of action, it is expected that suchpolypeptides act by binding to ligands of ALK1 and inhibiting theability of these ligands to interact with ALK1 as well as otherreceptors. In certain embodiments, an ALK1 ECD polypeptide comprises anamino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or100% identical to the sequence of amino acids 22-118 of the human ALK1sequence of SEQ ID NO:1. An ALK1 ECD polypeptide may be used as a smallmonomeric protein or in a dimerized form (e.g., expressed as a fusionprotein), particularly for local administration into tissues such as theeye. An ALK1 ECD may also be fused to a second polypeptide portion toprovide improved properties, such as an increased half-life or greaterease of production or purification. Fusions to an Fc portion of animmunoglobulin or linkage to a polyoxyethylene moiety (e.g.,polyethylene glycol) may be particularly useful to increase the serumhalf-life of the ALK1 ECD polypeptide in systemic administration (e.g.,intravenous, intraarterial and intra-peritoneal administration). Asdemonstrated herein, a systemically administered ALK1-Fc polypeptide hasa potent anti-angiogenic effect in the eye and also provides positiveeffects in murine models of rheumatoid arthritis and multiple myeloma.In certain embodiments, an ALK1-Fc fusion protein comprises apolypeptide having an amino acid sequence that is at least 70%, 80%,90%, 95%, 97%, 99% or 100% identical to the sequence of amino acids22-118 of SEQ ID NO:1, which polypeptide is fused, either with orwithout an intervening linker, to an Fc portion of an immunoglobulin,and wherein the ALK1-Fc fusion protein binds to GDF5, GDF7 and BMP9 witha K_(D) of less than 1×10⁻⁷ M and binds to TGFβ-1 with a K_(D) ofgreater than 1×10⁻⁶. An Fc portion may be selected so as to beappropriate to the organism. Optionally, the Fc portion is an Fc portionof a human IgG1. In a preferred embodiment, the ALK1-Fc fusion proteincomprises amino acids 22-118 of SEQ ID NO:1. Optionally, the ALK1-Fcfusion protein comprises the amino acid sequence of SEQ ID NO: 3.Optionally, the ALK1-Fc fusion protein is the protein produced byexpression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line,particularly a Chinese Hamster Ovary (CHO) cell line. ALK1-ECDpolypeptides may be formulated as a pharmaceutical preparation that issubstantially pyrogen free. The pharmaceutical preparation may beprepared for systemic delivery (e.g., intravenous, intraarterial orsubcutaneous delivery) or local delivery (e.g., to the eye).

In certain aspects, the disclosure provides methods for inhibitingangiogenesis in a mammal by administering any of the ALK1 ECDpolypeptides described generally or specifically herein. In oneembodiment, a method comprises administering to the mammal an effectiveamount of an ALK1-Fc fusion protein, wherein the ALK1 Fc fusion proteincomprises a polypeptide having an amino acid sequence that is at least90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1,which polypeptide is fused to an Fc portion of an immunoglobulin, andwherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K_(D) ofgreater than 1×10⁻⁶. Optionally, the ALK1-Fc fusion protein binds to oneor more ALK1 ligands selected from the group consisting of: GDF5, GDF6,GDF7, BMP9 and BMP10. Optionally, the ALK1-Fc fusion protein has asequence of SEQ ID NO:3. The ALK1 ECD polypeptide may be deliveredlocally (e.g., to the eye) or systemically (e.g., intravenously,intraarterially or subcutaneously). In a particular embodiment, thedisclosure provides a method for inhibiting angiogenesis in the eye of amammal by administering an ALK1-Fc protein to the mammal at a locationdistal to the eye, e.g. by systemic administration.

In certain aspects, the disclosure provides antibodies that bind toALK1, particularly an epitope situated in the extracellular domain,amino acids 22-118 of SEQ ID NO:1, and inhibit the binding of ALK1 to atleast one ALK1 ligand selected from the group consisting of: GDF5, GDF6,GDF7, BMP9 and BMP10. Based on the affinity of these ligands for ALK1,an antibody may bind with a K_(D) of less than 5×10⁻⁸ M, and optionallybetween 5×10⁻⁸ and 1×10⁻¹⁰. An antibody with affinity within this rangewould be expected to inhibit signaling by one or more of GDF5, 6 and 7while having less effect on signaling by BMP9 and 10. Such an antibodypreferably inhibits angiogenesis stimulated by at least one ALK1 ligandselected from the group consisting of: GDF5, GDF6 and GDF7. While notwishing to be bound to a particular mechanism, it is expected that suchantibodies will act by inhibiting ALK1 activity directly, which shouldbe contrasted to the activity of an ALK1-Fc fusion protein, which isexpected to inhibit the activity of ALK1 ligands. An anti-ALK1 antibodyis not expected to interfere with the ability of GDF5, GDF6, GDF7, BMP9or BMP10 to signal through alternative receptor systems, such as theBMPR1a, BMPR1b and BMPRII complexes. However, an anti-ALK1 antibody isexpected to interfere with the ability of low affinity ligands for ALK1(e.g., TGF-β, which is generally recognized as triggering significantsignaling events through ALK-1 even though binding is relatively weak)to signal through ALK1, even though an ALK1 ECD may not bind to orinhibit such low affinity ligands. An antibody may bind to the ALK1polypeptide with a K_(D) of less than 1×10⁻¹⁰ M. An antibody withaffinity within this range would be expected to inhibit signaling byBMP9 or 10. Such an antibody preferably inhibits binding of BMP9 andBMP10 to ALK1. Notably, based on the data disclosed herein, an antibodythat binds relatively poorly to ALK1 may inhibit TGFβ binding to ALK1while failing to inhibit the tighter binding ligands such as GDF5 orBMP9. The antibodies described herein are preferably recombinantantibodies, meaning an antibody expressed from a nucleic acid that hasbeen constructed using the techniques of molecular biology, such as ahumanized antibody or a fully human antibody developed from a singlechain antibody. Fv, Fab and single chain antibodies are also includedwithin the term “recombinant antibody”. Antibodies may also bepolyclonal or non-recombinant monoclonal antibodies (including human ormurine forms, as well as human antibodies obtained from transgenicmice). Antibodies and ALK1-ECD polypeptides may be formulated as apharmaceutical preparation that is substantially pyrogen free. Thepharmaceutical preparation may be prepared for systemic delivery (e.g.,intravenous, intraarterial or subcutaneous delivery) or local delivery(e.g., to the eye).

In certain aspects, the disclosure provides methods for inhibitingangiogenesis in a mammal by administering to the mammal an effectiveamount of an antibody that binds to an ALK1 polypeptide, describedherein either generally or specifically. An antibody useful for thispurpose may bind to the extracellular domain of ALK1 (e.g., bind to apolypeptide consisting of amino acids 22-118 of SEQ ID NO:1) or anotherportion of ALK1. The antibody may bind to a polypeptide consisting ofamino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at leastone ALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7,BMP9 and BMP10. The antibody may bind to the ALK1 polypeptide with aK_(D) of less than 5×10⁻⁸ M, and optionally between 5×10⁻⁸ and 1×10⁻¹⁰.The antibody may inhibit angiogenesis stimulated by at least one ALK1ligand selected from the group consisting of: GDF5, GDF6 and GDF7. Anantibody that selectively inhibits signaling mediated by GDF5, 6 or 7relative to signaling by BMP9 or 10 may be used as a selective inhibitorof angiogenesis that occurs in tissues where GDF5, 6 or 7 are localized:primarily bone or joints. The antibody may bind to the ALK1 polypeptidewith a K_(D) of less than 1×10⁻¹⁰ M. The antibody may inhibit thebinding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is selectedfrom the group consisting of: BMP9 and BMP10. The anti-ALK1 antibody maybe delivered locally (e.g., to the eye) or systemically (e.g.,intravenously, intraarterially or subcutaneously). In a particularembodiment, the disclosure provides a method for inhibiting angiogenesisin the eye of a mammal by administering an anti-ALK1 antibody. Inanother particular embodiment, the disclosure provides a method fortreating patients with multiple myeloma. In a particular embodiment, thedisclosure provides a method for inhibiting angiogenesis in disordersthat are associated with pathological angiogenesis as a consequence ofmultiple pro-angiogenic factors, such as VEGF, PDGF and/or FGF.

In certain aspects, the disclosure provides antibodies that bind to anALK1 ligand disclosed herein and inhibit the binding of the ALK1 ligandto ALK1. While not wishing to be bound to any particular mechanism, itis expected that antibodies that bind to ALK1 ligands will have effectsthat are similar in nature to ALK1 ECD polypeptides, because both typesof agent bind to the ligands rather than the receptor itself. In certainembodiments, the antibody binds to a ligand selected from the groupconsisting of GDF5, GDF6 and GDF7. The antibody may bind to the ALK1ligand with a K_(D) of less than 5×10⁻⁸ M. The antibody may be selectedfor inhibition of angiogenesis stimulated by the ALK1 ligand. A CAMassay is an appropriate assay system for selection of desirableantibodies. Such antibodies are preferably recombinant antibodies, andmay be formulated as a pharmaceutical preparation that is substantiallypyrogen free. The pharmaceutical preparation may be prepared forsystemic delivery (e.g., intravenous, intraarterial or subcutaneousdelivery) or local delivery (e.g., to the eye).

In certain aspects, the disclosure provides antibodies that bind to anALK1 ligand and inhibit the binding of the ALK1 ligand to ALK1, whereinthe ALK1 ligand is selected from the group consisting of BMP9 and BMP10.The antibody may bind to the ALK1 ligand with a K_(D) of less than1×10⁻¹⁰ M. Such antibodies are preferably recombinant antibodies, andmay be formulated as a pharmaceutical preparation that is substantiallypyrogen free. The pharmaceutical preparation may be prepared forsystemic delivery (e.g., intravenous, intraarterial or subcutaneousdelivery) or local delivery (e.g., to the eye).

In certain aspects, the disclosure provides methods for inhibitingangiogenesis in a mammal, the method comprising, administering to themammal an effective amount of an antibody that binds to an ALK1 ligandand inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1ligand is selected from the group consisting of GDF5, GDF6, GDF7, BMP9and BMP10. The antibody may inhibit angiogenesis stimulated by at leastone ALK1 ligand selected from the group consisting of: GDF5, GDF6 andGDF7.

Members of the BMP/GDF family, including BMP9, BMP10, GDF5, GDF6 andGDF7 bind to a type I and a type II receptor in order to form afunctional signaling complex. The binding sites for these receptors aredifferent. Accordingly, in certain embodiments, an antibody that bindsto an ALK1 ligand and inhibits the ligand to ALK1 is an antibody thatbinds at or near the type I receptor binding site of the ligand.

In certain aspects, the disclosure provides methods for inhibitingangiogenesis in a mammal by administering other inhibitors of the ALK1signaling system disclosed herein. Such inhibitors may include nucleicacids (e.g., antisense or RNAi constructs) that decrease the productionof ALK1, GDF5, GDF6, GDF7, BMP9 or BMP10). A variety of affinity bindingreagents can also be used, such as aptamers, random peptides, proteinscaffolds that can be modified to allow binding to selected targets(examples of such scaffolds include anticalins and FNIII domains); ineach case, an affinity binding reagent would be selected for the abilityto disrupt the ALK1 regulatory system disclosed herein, either bydisrupting the ALK1-ligand interaction or by inhibiting the signalingthat occurs after binding.

In a further embodiment, the disclosure describes the role of DAN as aregulator of the ALK1 regulatory system. As shown herein, DAN binds tothe GDF5 group of ligands but fails to bind to the BMP9 group ofligands. Thus, DAN is expected to inhibit angiogenesis mediated by GDF5,GDF6 or GDF7 but not angiogenesis mediated by BMP9 or BMP10. DAN maytherefore be used as a selective agent for inhibiting angiogenesis inthe bone or joints, where the GDF5 group of proteins is primarilyexpressed. Thus, in certain embodiments the disclosure provides DANproteins for use as anti-angiogenic agents in the context of bone orjoint angiogenesis, including rheumatoid arthritis and cancers thatinvolve the bone or joints (e.g., multiple myeloma and bone metastases).A DAN protein will generally bind to one or more ALK1 ligands selectedfrom the group consisting of: GDF5, GDF6 and GDF7, while havingrelatively poor binding to BMP9 or BMP10. A DAN protein may comprise anamino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or100% identical to the sequence of amino acids corresponding to aminoacids 17-180 of SEQ ID NO:10 (mature human DAN) or amino acids 21-125 ofSEQ ID NO:10 (conserved cysteine knot domain of DAN). A DAN protein mayalso be encoded by a nucleic acid that comprises a sequence thecomplement of which hybridizes under stringent hybridization conditionsto nucleotides 153-467 of SEQ ID NO:11 or a variant of nucleotides153-467 of SEQ ID NO:11 that has the same coding sequence (a “silent”variant, such as a variant containing one or more alterations at awobble position in the triplet code), or to nucleotides 93-635 of SEQ IDNO:11 or a silent variant thereof. In certain aspects, the DAN proteinis a fusion protein, such as an Fc fusion protein. While DAN is expectedto be particularly useful for the inhibition of angiogenesis in bone andjoints (including tumors located in the bone or joints, such as multiplemyeloma and bone metastases), it may also be useful in other contexts,such as in a tumor located elsewhere, or in the eye.

In certain aspects, the disclosure provides methods for treatingrheumatoid arthritis in a mammal, the method comprising, administeringto a mammal that has rheumatoid arthritis an effective amount of anagent selected from the group consisting of: an ALK1 ECD protein; anantibody that binds to an ALK1 ligand and inhibits the binding of theALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the groupconsisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that bindsto an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1and inhibits the binding of at least one ALK1 ligand selected from thegroup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and a DANpolypeptide.

In certain aspects the disclosure provides methods for treating a tumorin a mammal. Such a method may comprise administering to a mammal thathas a tumor an effective amount of an agent selected from the groupconsisting of: an ALK1 ECD protein; an antibody that binds to an ALK1ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein theALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7,BMP9 and BMP10; an antibody that binds to an ALK1 polypeptide consistingof amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of atleast one ALK1 ligand selected from the group consisting of: GDF5, GDF6,GDF7, BMP9 and BMP10; and a DAN polypeptide. A method may furthercomprise administering a second agent that inhibits angiogenesis. Atumor may be a tumor that is associated with bone, such as a leukemia, abone marrow tumor, a multiple myeloma or bone metastases, such as thosecommonly associated with breast or prostate cancer. A tumor may also beone that utilizes multiple pro-angiogenic factors, such as a tumor thatis resistant to anti-VEGF therapy.

In certain aspects the disclosure provides ophthalmic formulations. Suchformulations may comprise an agent selected from the group consistingof: an ALK1 ECD protein; an antibody that binds to an ALK1 ligand andinhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligandis selected from the group consisting of GDF5, GDF6, GDF7, BMP9 andBMP10; an antibody that binds to an ALK1 polypeptide consisting of aminoacids 22-118 of SEQ ID NO:1 and inhibits the binding of at least oneALK1 ligand selected from the group consisting of: GDF5, GDF6, GDF7,BMP9 and BMP10; and a DAN polypeptide.

In certain aspects, the disclosure provides methods for treating anangiogenesis related disease of the eye. Such methods may compriseadministering systemically or to said eye a pharmaceutical formulationcomprising: an effective amount of an agent selected from the groupconsisting of: an ALK1 ECD protein; an antibody that binds to an ALK1ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein theALK1 ligand is selected from the group consisting of GDF5, GDF6, GDF7,BMP9 and BMP10; an antibody that binds to an ALK1 polypeptide consistingof amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of atleast one ALK1 ligand selected from the group consisting of: GDF5, GDF6,GDF7, BMP9 and BMP10; and a DAN polypeptide.

In each instance, an agent described herein may be administered inconjunction with a second agent that inhibits angiogenesis. Where it isdesirable to inhibit angiogenesis of a tumor, the agent may beadministered in conjunction with a second agent that has an anti-cancereffect, such as a chemotherapeutic agent or a biologic anti-canceragent.

The disclosure also provides an ophthalmic pharmaceutical formulationcomprising an ALK1-Fc fusion protein having an amino acid sequence thatis at least 97% identical to the sequence of amino acids 22-118 of SEQID NO:1, which polypeptide is fused to an Fc portion of animmunoglobulin, and wherein the ALK1-Fc fusion protein binds to GDF5,GDF7 and BMP9 with a K_(D) of less than 1×10⁻⁷ M and binds to TGFβ-1with a K_(D) of greater than 1×10⁻⁶. In one embodiment, the fusionprotein has the sequence of SEQ ID NO: 3. In one embodiment, the Fcportion is from human IgG1. In one embodiment, the fusion protein isproduced by expression of the nucleic acid of SEQ ID NO:4 in a mammaliancell line. In one embodiment, the cell line is Chinese Hamster Ovarycell line. The formulation may further comprise one or more of thefollowing medicaments: pegaptanib, ranibizumab, or a glucocorticoid. Inone embodiment, the formulation is substantially pyrogen free.

The application also provides for an ophthalmic pharmaceuticalformulation comprising an antibody that binds to an ALK1 polypeptideconsisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the bindingof at least one ALK1 ligand selected from the group consisting of: GDF5,GDF6, GDF7, BMP9 and BMP10. In one embodiment, the antibody inhibitsangiogenesis stimulated by at least one ALK1 ligand selected from thegroup consisting of: GDF5, GDF6 and GDF7. In one embodiment, theantibody binds to the ALK1 polypeptide with a K_(D) of less than 5×10⁻⁸M. In another embodiment, the antibody binds to the ALK1 polypeptidewith a K_(D) of less than 1×10⁻¹⁰ M. In one embodiment, the antibodyinhibits angiogenesis stimulated by GDF5, GDF6, GDF7, BMP9, or BMP10.The formulation may further comprise one or more of the followingmedicaments: pegaptanib, ranibizumab, or a glucocorticoid. In oneembodiment, the formulation is substantially pyrogen free.

In certain aspects, the disclosure provides for an ophthalmicpharmaceutical formulation comprising an antibody that binds to an ALK1ligand disclosed herein and inhibits the binding of the ALK1 ligand toALK1. In certain embodiments, the antibody binds to a ligand selectedfrom the group consisting of GDF5, GDF6 and GDF7. The antibody may bindto the ALK1 ligand with a K_(D) of less than 5×10⁻⁸ M. The antibody maybe selected for inhibition of angiogenesis stimulated by the ALK1ligand. A CAM assay is an appropriate assay system for selection ofdesirable antibodies. Such antibodies are preferably recombinantantibodies. The formulation may further comprise one or more of thefollowing medicaments: pegaptanib, ranibizumab, or a glucocorticoid. Inone embodiment, the formulation is substantially pyrogen free.

The application also provides methods of treating an angiogenesisrelated disease of the eye comprising administering to said eye anophthalmic pharmaceutical formulation comprising an ALK1-Fc fusionprotein comprising: a polypeptide having an amino acid sequence that isat least 97% identical to the sequence of amino acids 22-118 of SEQ IDNO:1, which polypeptide is fused to an Fc portion of an immunoglobulin,and wherein the ALK1-Fc fusion protein binds to GDF5, GDF7 and BMP9 witha K_(D) of less than 1×10⁻⁷ M and binds to TGFβ-1 with a K_(D) ofgreater than 1×10⁻⁶. In one embodiment, the fusion protein has thesequence of SEQ ID NO: 3. In one embodiment, the Fc portion is fromhuman IgG1. In one embodiment, the fusion protein is produced byexpression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line.In one embodiment, the cell line is Chinese Hamster Ovary cell line. Theformulation may further comprise one or more of the followingmedicaments: pegaptanib, ranibizumab, or a glucocorticoid. In oneembodiment, the formulation is substantially pyrogen free.

The application also provides methods of treating an angiogenesisrelated disease of the eye comprising administering to said eye anophthalmic pharmaceutical formulation comprising an antibody that bindsto an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1and inhibits the binding of at least one ALK1 ligand selected from thegroup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. In oneembodiment, the antibody inhibits angiogenesis stimulated by at leastone ALK1 ligand selected from the group consisting of: GDF5, GDF6 andGDF7. In one embodiment, the antibody binds to the ALK1 polypeptide witha K_(D) of less than 5×10⁻⁸ M. In another embodiment, the antibody bindsto the ALK1 polypeptide with a K_(D) of less than 1×10⁻¹⁰ M. In oneembodiment, the antibody inhibits angiogenesis stimulated by GDF5, GDF6,GDF7, BMP9, or BMP10. The formulation may further comprise one or moreof the following medicaments: pegaptanib, ranibizumab, or aglucocorticoid. In one embodiment, the formulation is substantiallypyrogen free.

In one embodiment of the disclosed methods, the angiogenesis relateddisease of the eye is selected from the group consisting of a tumor, atumor that is resistant to anti-VEGF therapy, a multiple myeloma tumor,a tumor that has metastasized to the bone, joint or bone inflammation,rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma, andretrolental fibroplasias.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence for the human Activin Like Kinase1, ALK1 (SEQ ID NO:1). Single underlining shows the predictedextracellular domain. Double underlining shows the intracellular domain.The signal peptide and the transmembrane domain are not underlined.

FIG. 2 shows the nucleic acid sequence of a human ALK1 cDNA (SEQ IDNO:2). The coding sequence is underlined. The portion encoding theextracellular domain is double underlined.

FIG. 3 shows an example of a fusion of the extracellular domain of humanALK1 to an Fc domain (SEQ ID NO:3). The hALK1-Fc protein includes aminoacids 22-120 of the human ALK1 protein, fused at the C-terminus to alinker (underlined) and an IgG1 Fc region.

FIG. 4 shows the nucleic acid sequence for expression of the hALK1-Fcpolypeptide of SEQ ID NO:3. The encoded amino acid sequence is alsoshown. The leader sequence is cleaved such that Asp 22 is the N-terminalamino acid of the secreted protein.

FIG. 5 shows the anti-angiogenic effect of murine ALK1-Fc (“RAP”) andhuman ALK1-Fc (“ACE”) in an endothelial cell tube forming assay. Allconcentrations of RAP and ACE reduced the level of tube formation inresponse to Endothelial Cell Growth Supplement (ECGF) to a greaterdegree than the positive control, Endostatin.

FIG. 6 shows the angiogenic effect of GDF7 in a chick chorioallantoicmembrane (CAM) assay. The GDF7 effect is comparable to that of VEGF.

FIG. 7 shows the anti-angiogenic effect of the human ALK1-Fc fusion inthe CAM assay. hALK1-Fc inhibits angiogenesis stimulated by VEGF, FGFand GDF7.

FIG. 8 shows comparative anti-angiogenic effects of murine ALK1-Fc(mALK1-Fc), hALK1-Fc, a commercially available anti-ALK1 monoclonalantibody (Anti-ALK1 mAb) and a commercially available, neutralizinganti-VEGF monoclonal antibody. The anti-angiogenic effect of the ALK1-Fcconstructs is comparable to the effects of the anti-VEGF antibody.

FIG. 9 shows the anti-angiogenic effects of hALK1-Fc and the anti-VEGFantibody in vivo. hALK1-Fc and anti-VEGF had comparable effects onangiogenesis in the eye as measured by the mouse corneal micropocketassay.

FIG. 10 shows the effects of mALK1-Fc in the murine collagen-inducedarthritis (CIA) model of rheumatoid arthritis. The graph shows meangroup arthritic scores determined during the 42 day observation periodin the collagen-induced male DBA/1 arthritic mice. RAP-041 is mALK1-Fc.Avastin™ is the anti-VEGF antibody bevacizumab.

DETAILED DESCRIPTION 1. Overview

ALK1 is a type I cell-surface receptor for the TGF-0 superfamily ofligands and is also known as ACVRL1 and ACVRLK1. ALK1 has beenimplicated as a receptor for TGF-β1, TGF-β3 and BMP-9 (Marchuk et al.,Hum Mol. Genet. 2003; Brown et al., J Biol. Chem. 2005 Jul. 1;280(26):25111-8).

In mice, loss-of-function mutations in ALK1 lead to a variety ofabnormalities in the developing vasculature (Oh et al., Proc. Natl.Acad. Sci. USA 2000, 97, 2626-2631; Urness et al., Nat. Genet. 2000, 26,328-331).

In humans, loss-of-function mutations in ALK1 are associated withhereditary hemorrhagic telangiectasia (HHT, or Osler-Rendu-Webersyndrome), in which patients develop arteriovenous malformations thatcreate direct flow (communication) from an artery to a vein(arteriovenous shunt), without an intervening capillary bed. Typicalsymptoms of patients with HHT include recurrent epistaxis,gastrointestinal hemorrhage, cutaneous and mucocutaneous telangiectases,and arteriovenous malformations (AVM) in the pulmonary, cerebral, orhepatic vasculature.

Recent publications from David et al. (Blood. 2007 Mar. 1;109(5):1953-61.) and Scharpfenecker et al. (J Cell Sci. 2007 Mar. 15;120(Pt 6):964-72) conclude that BMP9 and BMP10 activate ALK1 inendothelial cells, and that the consequence of this activation is toinhibit endothelial cell proliferation and migration. These effects aredirectly opposed to those of pro-angiogenic factors such as VEGF. Thus,these publications conclude that BMP9 and BMP10 are themselvesanti-angiogenic factors, and further, that ALK1 activation has ananti-angiogenic effect. By contrast, the present disclosure demonstratesthat antagonists, rather than agonists, of BMP9 and BMP10 haveanti-angiogenic effects.

The disclosure relates to the discovery that polypeptides comprising aportion of the extracellular domain of ALK1 (“ALK1 ECD polypeptides”)may be used to inhibit angiogenesis in vivo, including VEGF-independentangiogenesis and angiogenesis that is mediated by multiple angiogenicfactors, including VEGF, FGF and PDGF. In part, the disclosure providesthe identity of physiological, high affinity ligands for ALK1 anddemonstrates that ALK1 ECD polypeptides inhibit angiogenesis. The datademonstrate that an ALK1 ECD polypeptide can exert an anti-angiogeniceffect even in the case where the ALK1 ECD polypeptide does not exhibitmeaningful binding to TGF-β1. Moreover, ALK1 ECD polypeptides inhibitangiogenesis that is stimulated by many different pro-angiogenicfactors, including VEGF, FGF, and GDF7. Thus, the disclosure provides adescription of an ALK1 regulatory system, in which ALK1 is a receptorfor the GDF5 group of ligands, which includes GDF6 and GDF7, and alsofor the BMP9 group of ligands, which includes BMP10, with differentaffinities for the two groups of ligands. Further, the disclosuredemonstrates that signaling mediated by ALK1 and the ligands describedabove is pro-angiogenic in vivo, and that inhibition of this regulatorysystem has a potent anti-angiogenic effect in vivo. Thus, in certainaspects, the disclosure provides antagonists of the ALK1 regulatorysystem, including antagonists of the receptor or one or more of theligands, for use in inhibiting angiogenesis, including bothVEGF-dependent angiogenesis and VEGF-independent angiogenesis. However,it should be noted that antibodies directed to ALK1 itself are expectedto have different effects from an ALK1 ECD polypeptide. Apan-neutralizing antibody against ALK1 (one that inhibits the binding ofall strong and weak ligands) would be expected to inhibit the signalingof such ligands through ALK1 but would not be expected to inhibit theability of such ligands to signal through other receptors (e.g., BMPR1a,BMPR1b, BMPRII in the case of GDF5-7 and BMP9-10 and TBRI and TBRII inthe case of TGFβ). On the other hand, an ALK1 ECD polypeptide would beexpected to inhibit all of the ligands that it binds to tightly,including, for a construct such as that shown in the Examples, GDF5-7and BMP9-10, but would not affect ligands that it binds to weakly, suchas TGF-β. So, while a pan-neutralizing antibody against ALK1 would blockBMP9 and TGF-β signaling through ALK1 it would not block BMP9 and TGF-βsignaling through another receptor, and while an ALK1 ECD polypeptidemay inhibit BMP9 signaling through all receptors (even receptors otherthan ALK1) it would not be expected to inhibit TGF-β signaling throughany receptor, even ALK1.

Proteins described herein are the human forms, unless otherwisespecified. Genbank references for the proteins are as follows: humanGDF5, CAA56874; human GDF6, AAH43222; human GDF7, NP_(—)878248; humanBMP9, Q9UK05; human BMP10, O95393; human DAN, BAA92265. ALK1 sequencesare set forth in FIGS. 1-5.

Human Dan amino acid sequence (SEQ ID NO: 10) (Genbank BAA92265):MLRVLVGAVL PAMLLAAPPP INKLALFPDK SAWCEAKNITQIVGHSGCEA KSIQNRACLG QCFSYSVPNT FPQSTESLVHCDSCMPAQSM WEIVTLECPG HEEVPRVDKL VEKILHCSCQACGKEPSHEG LSVYVQGEDG PGSQPGTHPH PHPHPHPGGQ TPEPEDPPGA PHTEEEGAED

The mature Dan protein is expected to correspond to amino acids 17-180.The conserved cysteine knot domain of Dan corresponds to amino acids21-125 (underlined).

Human Dan cDNA sequence (SEQ ID NO: 11) (Genbank BC012037):gccgagcctc ctggggcgcc cgggcccgcg acccccgcacccagctccgc aggaccggcg ggcgcgcgcg ggctctggaggccacgggca tgatgcttcg ggtcctggtg ggggctgtcctccctgccat gctactggct gccccaccac ccatcaacaagctggcactg ttcccagata agagtgcctg gtgcgaagccaagaacatca cccagatcgt gggccacagc ggctgtgaggccaagtccat ccagaacagg gcgtgcctag gacagtgcttcagctacagc gtccccaaca ccttcccaca gtccacagagtccctggttc actgtgactc ctgcatgcca gcccagtccatgtgggagat tgtgacgctg gagtgcccgg gccacgaggaggtgcccagg gtggacaagc tggtggagaa gatcctgcactgtagctgcc aggcctgcgg caaggagcct agtcacgaggggctgagcgt ctatgtgcag ggcgaggacg ggccgggatcccagcccggc acccaccctc acccccatcc ccacccccatcctggcgggc agacccctga gcccgaggac ccccctggggccccccacac agaggaagag ggggctgagg actgaggcccccccaactct tcctcccctc tcatccccct gtggaatgttgggtctcact ctctggggaa gtcaggggag aagctgaagcccccctttgg cactggatgg acttggcttc agactcggacttgaatgctg cccggttgcc atggagatct gaaggggcggggttagagcc aagctgcaca atttaatata ttcaagagtggggggaggaa gcagaggtct tcagggctct ttttttggggggggggtggt ctcttcctgt ctggcttcta gagatgtgcctgtgggaggg ggaggaagtt ggctgagcca ttgagtgctgggggaggcca tccaagatgg catgaatcgg gctaaggtccctgggggtgc agatggtact gctgaggtcc cgggcttagtgtgagcatct tgccagcctc aggcttgagg gagggctgggctagaaagac cactggcaga aacaggaggc tccggccccacaggtttccc caaggcctct caccccactt cccatctccagggaagcgtc gccccagtgg cactgaagtg gccctccctcagcggagggg tttgggagtc aggcctgggc aggaccctgctgactcgtgg cgcgggagct gggagccagg ctctccgggcctttctctgg cttccttggc ttgcctggtg ggggaaggggaggaggggaa gaaggaaagg gaagagtctt ccaaggccagaaggaggggg acaacccccc aagaccatcc ctgaagacgagcatccccct cctctccctg ttagaaatgt tagtgccccgcactgtgccc caagttctag gccccccaga aagctgtcagagccggccgc cttctcccct ctcccaggga tgctctttgtaaatatcgga tgggtgtggg agtgaggggt tacctccctcgccccaaggt tccagaggcc ctaggcggga tgggctcgctgaacctcgag gaactccagg acgaggagga catgggacttgcgtggacag tcagggttca cttgggctct ctctagctccccaattctgc ctgcctcctc cctcccagct gcactttaaccctagaaggt ggggacctgg ggggagggac agggcaggcgggcccatgaa gaaagcccct cgttgcccag cactgtctgcgtctgctctt ctgtgcccag ggtggctgcc agcccactgcctcctgcctg gggtggcctg gccctcctgg ctgttgcgacgcgggcttct ggagcttgtc accattggac agtctccctgatggaccctc agtcttctca tgaataaatt ccttcaacgccaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa

The coding sequence for DAN precursor corresponds to nucleic acids93-635. The coding sequence for the mature DAN protein corresponds tonucleic acids 141-632. The coding sequence for the conserved cysteineknot portion of DAN corresponds to nucleic acids 153-467.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussed inthe specification, to provide additional guidance to the practitioner indescribing the compositions and methods disclosed herein and how to makeand use them. The scope or meaning of any use of a term will be apparentfrom the specific context in which the term is used.

2. Soluble ALK1 Polypeptides

Naturally occurring ALK1 proteins are transmembrane proteins, with aportion of the protein positioned outside the cell (the extracelluarportion) and a portion of the protein positioned inside the cell (theintracellular portion). Aspects of the present disclosure encompasspolypeptides comprising a portion of the extracellular domain of ALK1.

In certain embodiments, the disclosure provides “ALK1 ECD polypeptides”.The term “ALK1 ECD polypeptide” is intended to refer to a polypeptideconsisting of or comprising an amino acid sequence of an extracellulardomain of a naturally occurring ALK1 polypeptide, either including orexcluding any signal sequence and sequence N-terminal to the signalsequence, or an amino acid sequence that is at least 33 percentidentical to an extracellular domain of a naturally occurring ALK1polypeptide, and optionally at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to the sequence of an extracellular domain of a naturallyoccurring ALK1 polypeptide, as exemplified by the cysteine knot regionof amino acids 34-95 of SEQ ID No:1 or the cysteine knot plus additionalamino acids at the N- and C-termini of the extracellular domain, such asamino acids 22-118 of SEQ ID No. 1. Likewise, an ALK1 ECD polypeptidemay comprise a polypeptide that is encoded by nucleotides 100-285 of SEQID NO:2, or silent variants thereof or nucleic acids that hybridize tothe complement thereof under stringent hybridization conditions(generally, such conditions are known in the art but may, for example,involve hybridization in 50% v/v formamide, 5×SSC, 2% w/v blockingagent, 0.1% N-lauroylsarcosine, 0.3% SDS at 65 C.° overnight and washingin, for example, SxSSC at about 65 C.°). Additionally, an ALK1 ECDpolypeptide may comprise a polypeptide that is encoded by nucleotides64-384 of SEQ ID NO:2, or silent variants thereof or nucleic acids thathybridize to the complement thereof under stringent hybridizationconditions (generally, such conditions are known in the art but may, forexample, involve hybridization in 50% v/v formamide, 5×SSC, 2% w/vblocking agent, 0.1% N-lauroylsarcosine, 0.3% SDS at 65 C.° overnightand washing in, for example, 5×SSC at about 65 C.°). The term “ALK1 ECDpolypeptide” accordingly encompasses isolated extracellular portions ofALK1 polypeptides, variants thereof (including variants that comprise,for example, no more than 2, 3, 4, 5 or 10 amino acid substitutions,additions or deletions in the sequence corresponding to amino acids22-118 of SEQ ID NO:1 and including variants that comprise no more than2, 3, 4, 5, or 10 amino acid substitutions, additions or deletions inthe sequence corresponding to amino acids 34-95 of SEQ ID NO:1),fragments thereof and fusion proteins comprising any of the preceding,but in each case preferably any of the foregoing ALK1 ECD polypeptideswill retain substantial affinity for one or more of GDF5, GDF6, GDF7,BMP9 or BMP10. The term “ALK1 ECD polypeptide” is explicitly intended toexclude any full-length, naturally occurring ALK1 polypeptide.Generally, an ALK1 ECD polypeptide will be designed to be soluble inaqueous solutions at biologically relevant temperatures, pH levels andosmolarity.

As described above, the disclosure provides ALK1 ECD polypeptidessharing a specified degree of sequence identity or similarity to anaturally occurring ALK1 polypeptide. To determine the percent identityof two amino acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). The amino acid residues at corresponding amino acid positionsare then compared. When a position in the first sequence is occupied bythe same amino acid residue as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid “identity” is equivalent to amino acid “homology”).The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

In one embodiment, the percent identity between two amino acid sequencesis determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available at http://www.gcg.com). In aspecific embodiment, the following parameters are used in the GAPprogram: either a Blosum 62 matrix or a PAM250 matrix, and a gap weightof 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or6. In yet another embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)) (available at http://www.gcg.com). Exemplary parameters includeusing a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified,percent identity between two amino acid sequences is to be determinedusing the GAP program using a Blosum 62 matrix, a GAP weight of 10 and alength weight of 3, and if such algorithm cannot compute the desiredpercent identity, a suitable alternative disclosed herein should beselected.

In another embodiment, the percent identity between two amino acidsequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment betweentwo amino acid sequences can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.,6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is presented in terms of percent identity. In oneembodiment, amino acid sequence identity is performed using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

In certain embodiments, ALK1 ECD polypeptides comprise an extracellularportion of a naturally occurring ALK1 protein such as a sequence of SEQID NO:1, and preferably a ligand binding portion of the ALK1extracellular domain. In certain embodiments, a soluble ALK1 polypeptidecomprises an amino acid sequence that is at least 60%, 70%, 80%, 85%,90%, 95%, 97% or 99% identical to an amino acid sequence of amino acids22-118 of the SEQ ID NO:1. In certain embodiments, a truncatedextracellular ALK1 polypeptide comprises at least 30, 40 or 50consecutive amino acids of an amino acid sequence of an extracellularportion of SEQ ID NO:1.

In preferred embodiments, an ALK1 ECD polypeptide binds to one or moreof GDF5, GDF6, GDF7, BMP9 and BMP10. Optionally the ALK1 polypeptidedoes not show substantial binding to TGF-131 or TGF-133. Binding may beassessed using purified proteins in solution or in a surface plasmonresonance system, such as a Biacore™ system. Preferred soluble ALK1polypeptides will exhibit an anti-angiogenic activity. Bioassays forangiogenesis inhibitory activity include the chick chorioallantoicmembrane (CAM) assay, the mouse corneal micropocket assay, an assay formeasuring the effect of administering isolated or synthesized proteinson implanted tumors. The CAM assay is described by O'Reilly, et al. in“Angiogenic Regulation of Metastatic Growth” Cell, vol. 79 (2), Oct. 1,1994, pp. 315-328. Briefly, 3 day old chicken embryos with intact yolksare separated from the egg and placed in a petri dish. After 3 days ofincubation, a methylcellulose disc containing the protein to be testedis applied to the CAM of individual embryos. After 48 hours ofincubation, the embryos and CAMs are observed to determine whetherendothelial growth has been inhibited. The mouse corneal micropocketassay involves implanting a growth factor-containing pellet, along withanother pellet containing the suspected endothelial growth inhibitor, inthe cornea of a mouse and observing the pattern of capillaries that areelaborated in the cornea. Other assays are described in the Examples.

ALK1 ECD polypeptides may be produced by removing the cytoplasmic tailand the transmembrane region of an ALK1 polypeptide. Alternatively, thetransmembrane domain may be inactivated by deletion, or by substitutionof the normally hydrophobic amino acid residues which comprise atransmembrane domain with hydrophilic ones. In either case, asubstantially hydrophilic hydropathy profile is created which willreduce lipid affinity and improve aqueous solubility. Deletion of thetransmembrane domain is preferred over substitution with hydrophilicamino acid residues because it avoids introducing potentiallyimmunogenic epitopes.

ALK1 ECD polypeptides may additionally include any number of well-knownleader sequences at the N-terminus. Such a sequence would allow thepeptides to be expressed and targeted to the secretion pathway in aeukaryotic system. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783(1992). Alternatively, a native ALK1 signal sequence may be used toeffect extrusion from the cell. Possible leader sequences includenative, tPa and honeybee mellitin leaders (SEQ ID Nos. 7-9,respectively). Processing of signal peptides may vary depending on theleader sequence chosen, the cell type used and culture conditions, amongother variables, and therefore actual N-terminal start sites for matureALK1 ECD polypeptides, including that of SEQ ID NO:5, may shift by 1-5amino acids in either the N-terminal or C-terminal direction.

In certain embodiments, the present disclosure contemplates specificmutations of the ALK1 polypeptides so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagines-X-serine) (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ALK1 polypeptide (for O-linked glycosylation sites). A varietyof amino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Another means of increasing thenumber of carbohydrate moieties on an ALK1 polypeptide is by chemical orenzymatic coupling of glycosides to the ALK1 polypeptide. Depending onthe coupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on an ALK1 polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the ALK1 polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ALK1 polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al. (1987) Meth.Enzymol. 138:350. The sequence of an ALK1 polypeptide may be adjusted,as appropriate, depending on the type of expression system used, asmammalian, yeast, insect and plant cells may all introduce differingglycosylation patterns that can be affected by the amino acid sequenceof the peptide. In general, ALK1 proteins for use in humans will beexpressed in a mammalian cell line that provides proper glycosylation,such as HEK293 or CHO cell lines, although other mammalian expressioncell lines, yeast cell lines with engineered glycosylation enzymes andinsect cells are expected to be useful as well.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ALK1 polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ALK1 polypeptide variants which can act as either agonistsor antagonist, or alternatively, which possess novel activities alltogether. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ALK1polypeptide variant may be screened for ability to bind to an ALK1ligand, to prevent binding of an ALK1 ligand to an ALK1 polypeptide orto interfere with signaling caused by an ALK1 ligand. The activity of anALK1 polypeptide or its variants may also be tested in a cell-based orin vivo assay, particularly any of the assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to an ALK1 ECD polypeptidecomprising an extracellular domain of a naturally occurring ALK1polypeptide. Likewise, mutagenesis can give rise to variants which haveserum half-lives dramatically different than the corresponding awild-type ALK1 ECD polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother processes which result in destruction of, or otherwise eliminationor inactivation of a native ALK1 ECD polypeptide. Such variants, and thegenes which encode them, can be utilized to alter ALK1 ECD polypeptidelevels by modulating the half-life of the ALK1 polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and can allow tighter control of recombinant ALK1 ECDpolypeptide levels within the patient. In an Fc fusion protein,mutations may be made in the linker (if any) and/or the Fc portion toalter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ALK1 polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ALK1polypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential ALK1 ECD variantscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then be ligated intoan appropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakuraet al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ALK1 polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ALK1 polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ALK1 polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include ALK1 ligand binding assays and ligand-mediated cellsignaling assays.

In certain embodiments, the ALK1 ECD polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ALK1 polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. As a result,the modified ALK1 ECD polypeptides may contain non-amino acid elements,such as polyethylene glycols, lipids, poly- or mono-saccharide, andphosphates. Effects of such non-amino acid elements on the functionalityof an ALK1 ECD polypeptide may be tested as described herein for otherALK1 ECD polypeptide variants. When an ALK1 ECD polypeptide is producedin cells by cleaving a nascent form of the ALK1 polypeptide,post-translational processing may also be important for correct foldingand/or function of the protein. Different cells (such as CHO, HeLa,MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of the ALK1polypeptides.

In certain aspects, functional variants or modified forms of the ALK1ECD polypeptides include fusion proteins having at least a portion ofthe ALK1 ECD polypeptides and one or more fusion domains. Well knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. A fusiondomain may be selected so as to confer a desired property. For example,some fusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ALK1 ECDpolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ALK1ECD polypeptide is fused with a domain that stabilizes the ALK1polypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains.

As a specific example, the present disclosure provides a fusion proteincomprising a soluble extracellular domain of ALK1 fused to an Fc domain(e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fey receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ALK1 ECD polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ALK1 ECD polypeptide. The ALK1 ECDpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

As used herein, the term, “immunoglobulin Fc region” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) andIgM (Igμ), may be used. The choice of appropriate immunoglobulin heavychain constant region is discussed in detail in U.S. Pat. Nos.5,541,087, and 5,726,044. The choice of particular immunoglobulin heavychain constant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH₃ domain of Fc γor the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613).

In certain embodiments, the present disclosure makes available isolatedand/or purified forms of the ALK1 ECD polypeptides, which are isolatedfrom, or otherwise substantially free of (e.g., at least 80%, 90%, 95%,97% or 99% free of), other proteins and/or other ALK1 ECD polypeptidespecies. ALK1 polypeptides will generally be produced by expression fromrecombinant nucleic acids.

In certain embodiments, the disclosure includes nucleic acids encodingsoluble ALK1 polypeptides comprising the coding sequence for anextracellular portion of an ALK1 proteins. In further embodiments, thisdisclosure also pertains to a host cell comprising such nucleic acids.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing the ALK1ECD polypeptides. It has been established that an ALK1-Fc fusion proteinset forth in SEQ ID NO:3 and expressed in CHO cells has potentanti-angiogenic activity.

DAN polypeptides, including variants of wild type DAN, and fusionproteins containing DAN proteins may be generated and characterized asdescribed above with respect to ALK1 ECD proteins.

3. Nucleic Acids Encoding ALK1 Polypeptides

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the ALK1 polypeptides (e.g., ALK1 ECDpolypeptides), including fragments, functional variants and fusionproteins disclosed herein. For example, SEQ ID NO: 2 encodes thenaturally occurring human ALK1 precursor polypeptide, while SEQ ID NO: 4encodes the precursor of an ALK1 extracellular domain fused to an IgG1Fc domain. The subject nucleic acids may be single-stranded or doublestranded. Such nucleic acids may be DNA or RNA molecules. These nucleicacids may be used, for example, in methods for making ALK1 polypeptidesor as direct therapeutic agents (e.g., in an antisense, RNAi or genetherapy approach).

In certain aspects, the subject nucleic acids encoding ALK1 polypeptidesare further understood to include nucleic acids that are variants of SEQID NO: 2 or 4. Variant nucleotide sequences include sequences thatdiffer by one or more nucleotide substitutions, additions or deletions,such as allelic variants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 2 or 4. One of ordinary skill in theart will appreciate that nucleic acid sequences complementary to SEQ IDNO: 2 or 4, and variants of SEQ ID NO: 2 or 4 are also within the scopeof this disclosure. In further embodiments, the nucleic acid sequencesof the disclosure can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 2 or 4, complementsequence of SEQ ID NO: 2 or 4, or fragments thereof. As discussed above,one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the disclosure provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 2 or 4 due to degeneracy in the genetic code are alsowithin the scope of the disclosure. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ALK1 polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ALK1 polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ALK1 polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., PhoS, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ALK1 polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Ban virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ALK1 polypeptides in CHO cells, such as a Pcmv-Script vector(Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad,Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will beapparent, the subject gene constructs can be used to cause expression ofthe subject ALK1 polypeptides in cells propagated in culture, e.g., toproduce proteins, including fusion proteins or variant proteins, forpurification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 2 or 4)for one or more of the subject ALK1 polypeptides. The host cell may beany prokaryotic or eukaryotic cell. For example, an ALK1 polypeptidedisclosed herein may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ALK1 polypeptides, including ALK1 ECDpolypeptides. For example, a host cell transfected with an expressionvector encoding an ALK1 polypeptide can be cultured under appropriateconditions to allow expression of the ALK1 polypeptide to occur. TheALK1 polypeptide may be secreted and isolated from a mixture of cellsand medium containing the ALK1 polypeptide. Alternatively, the ALK1polypeptide may be retained cytoplasmically or in a membrane fractionand the cells harvested, lysed and the protein isolated. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The subject ALK1 polypeptides can beisolated from cell culture medium, host cells, or both, using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, immunoaffinity purification with antibodies specificfor particular epitopes of the ALK1 polypeptides and affinitypurification with an agent that binds to a domain fused to the ALK1polypeptide (e.g., a protein A column may be used to purify an ALK1-Fcfusion). In a preferred embodiment, the ALK1 polypeptide is a fusionprotein containing a domain which facilitates its purification. In apreferred embodiment, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ALK1polypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ALK1 polypeptide (e.g., see Hochuliet al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

Examples of categories of nucleic acid compounds that are antagonists ofALK1, BMP9, BMP10, GDF5, GDF6 or GDF7 include antisense nucleic acids,RNAi constructs and catalytic nucleic acid constructs. A nucleic acidcompound may be single or double stranded. A double stranded compoundmay also include regions of overhang or non-complementarity, where oneor the other of the strands is single stranded. A single strandedcompound may include regions of self-complementarity, meaning that thecompound forms a so-called “hairpin” or “stem-loop”structure, with aregion of double helical structure. A nucleic acid compound may comprisea nucleotide sequence that is complementary to a region consisting of nomore than 1000, no more than 500, no more than 250, no more than 100 orno more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-lengthALK1 nucleic acid sequence or ligand nucleic acid sequence. The regionof complementarity will preferably be at least 8 nucleotides, andoptionally at least 10 or at least 15 nucleotides, and optionallybetween 15 and 25 nucleotides. A region of complementarity may fallwithin an intron, a coding sequence or a noncoding sequence of thetarget transcript, such as the coding sequence portion. Generally, anucleic acid compound will have a length of about 8 to about 500nucleotides or base pairs in length, and optionally the length will beabout 14 to about 50 nucleotides. A nucleic acid may be a DNA(particularly for use as an antisense), RNA or RNA:DNA hybrid. Any onestrand may include a mixture of DNA and RNA, as well as modified formsthat cannot readily be classified as either DNA or RNA. Likewise, adouble stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any onestrand may also include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. A nucleicacid compound may include any of a variety of modifications, includingone or modifications to the backbone (the sugar-phosphate portion in anatural nucleic acid, including internucleotide linkages) or the baseportion (the purine or pyrimidine portion of a natural nucleic acid). Anantisense nucleic acid compound will preferably have a length of about15 to about 30 nucleotides and will often contain one or moremodifications to improve characteristics such as stability in the serum,in a cell or in a place where the compound is likely to be delivered,such as the stomach in the case of orally delivered compounds and thelung for inhaled compounds. In the case of an RNAi construct, the strandcomplementary to the target transcript will generally be RNA ormodifications thereof. The other strand may be RNA, DNA or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will preferably have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90% or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,angiogenesis.

Nucleic acids encoding DAN polypeptides, including variants of wild typeDAN, and those encoding fusion proteins containing DAN proteins may begenerated and characterized as described above with respect to nucleicacids encoding ALK1 ECD proteins.

4. Antibodies

Another aspect of the disclosure pertains to an antibody reactive withan extracellular portion of an ALK1 polypeptide, preferably antibodiesthat are specifically reactive with ALK1 polypeptide. In a preferredembodiment, such antibody may interfere with ALK1 binding to a ligandsuch as GDF5, GDF6, GDF7 BMP-9 or BMP-10—it will be understood that anantibody against a ligand of ALK1 should bind to the mature, processedform of the relevant protein. The disclosure also provides antibodiesthat bind to GDF5, GDF6, GDF7, BMP9 and/or BMP10 and inhibit ALK1binding to such ligands. Preferred antibodies will exhibit ananti-angiogenic activity in a bioassay, such as a CAM assay or cornealmicropocket assay (see above).

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments or domains of immunoglobulins which are reactive with aselected antigen. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility and/or interactionwith a specific epitope of interest. Thus, the term includes segments ofproteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The term antibody alsoincludes polyclonal, monoclonal, or other purified preparations ofantibodies and recombinant antibodies. The term “recombinant antibody”,means an antibody, or antigen binding domain of an immunoglobulin,expressed from a nucleic acid that has been constructed using thetechniques of molecular biology, such as a humanized antibody or a fullyhuman antibody developed from a single chain antibody. Single domain andsingle chain antibodies are also included within the term “recombinantantibody”.

Antibodies may be generated by any of the various methods known in theart, including administration of antigen to an animal, administration ofantigen to an animal that carries human immunoglobulin genes, orscreening with an antigen against a library of antibodies (often singlechain antibodies or antibody domains). Once antigen binding activity isdetected, the relevant portions of the protein may be grafted into otherantibody frameworks, including full-length IgG frameworks. For example,by using immunogens derived from an ALK1 polypeptide or an ALK1 ligand,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the peptide (e.g., a ALK1 polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusionprotein). Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion (preferably an extracellular portion)of an ALK1 polypeptide can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassays can beused with the immunogen as antigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anALK1 polypeptide, anti-ALK1 antisera can be obtained and, if desired,polyclonal anti-ALK1 antibodies can be isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian ALK1polypeptide of the present disclosure and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectALK1 polypeptides. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner asdescribed above for whole antibodies. For example, F(ab)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present disclosure is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ALK1 polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and is able to be detected, (e.g.,the label can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the disclosure is arecombinant antibody, particularly a humanized monoclonal antibody or afully human recombinant antibody.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g. an ALK1 polypeptide or an ALK1 ligand) and other antigensthat are not of interest that the antibody is useful for, at minimum,detecting the presence of the antigen of interest in a particular typeof biological sample. In certain methods employing the antibody, ahigher degree of specificity in binding may be desirable. For example,an antibody for use in detecting a low abundance protein of interest inthe presence of one or more very high abundance protein that are not ofinterest may perform better if it has a higher degree of selectivitybetween the antigen of interest and other cross-reactants. Monoclonalantibodies generally have a greater tendency (as compared to polyclonalantibodies) to discriminate effectively between the desired antigens andcross-reacting polypeptides. In addition, an antibody that is effectiveat selectively identifying an antigen of interest in one type ofbiological sample (e.g. a stool sample) may not be as effective forselectively identifying the same antigen in a different type ofbiological sample (e.g. a blood sample). Likewise, an antibody that iseffective at identifying an antigen of interest in a purified proteinpreparation that is devoid of other biological contaminants may not beas effective at identifying an antigen of interest in a crude biologicalsample, such as a blood or urine sample. Accordingly, in preferredembodiments, the application provides antibodies that have demonstratedspecificity for an antigen of interest in a sample type that is likelyto be the sample type of choice for use of the antibody.

One characteristic that influences the specificity of anantibody:antigen interaction is the affinity of the antibody for theantigen. Although the desired specificity may be reached with a range ofdifferent affinities, generally preferred antibodies will have anaffinity (a dissociation constant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ orless. Given the apparently low binding affinity of TGFβ for ALK1, it isexpected that many anti-ALK1 antibodies will inhibit TGFβ binding.However, the GDF5, 6, 7 group of ligands bind with a K_(D) ofapproximately 5×10⁻⁸ M and the BMP9, 10 ligands bind with a K_(D) ofapproximately 1×10⁻¹⁰ M. Thus, antibodies of appropriate affinity may beselected to interfere with the signaling activities of these ligands.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, an antibody to be used for certaintherapeutic purposes will preferably be able to target a particular celltype. Accordingly, to obtain antibodies of this type, it may bedesirable to screen for antibodies that bind to cells that express theantigen of interest (e.g. by fluorescence activated cell sorting).Likewise, if an antibody is to be used for binding an antigen insolution, it may be desirable to test solution binding. A variety ofdifferent techniques are available for testing antibody:antigeninteractions to identify particularly desirable antibodies. Suchtechniques include ELISAs, surface plasmon resonance binding assays(e.g. the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwichassays (e.g. the paramagnetic bead system of IGEN International, Inc.,Gaithersburg, Md.), western blots, immunoprecipitation assays andimmunohistochemistry.

5. Alterations in Antibodies and Fc-Fusion Proteins

The application further provides antibodies, ALK1-Fc fusion proteins andDAN-Fc fusion proteins with engineered or variant Fc regions. Suchantibodies and Fc fusion proteins may be useful, for example, inmodulating effector functions, such as, antigen-dependent cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC). Additionally, themodifications may improve the stability of the antibodies and Fc fusionproteins. Amino acid sequence variants of the antibodies and Fc fusionproteins are prepared by introducing appropriate nucleotide changes intothe DNA, or by peptide synthesis. Such variants include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibodies and Fc fusion proteinsdisclosed herein. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the antibodiesand Fc fusion proteins, such as changing the number or position ofglycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may beproduced by introducing changes in the amino acid sequence, including,but are not limited to, the Ala-Ala mutation described by Bluestone etal. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 CellImmunol 200; 16-26). Thus in certain embodiments, antibodies and Fcfusion proteins of the disclosure with mutations within the constantregion including the Ala-Ala mutation may be used to reduce or abolisheffector function. According to these embodiments, antibodies and Fcfusion proteins may comprise a mutation to an alanine at position 234 ora mutation to an alanine at position 235, or a combination thereof. Inone embodiment, the antibody or Fc fusion protein comprises an IgG4framework, wherein the Ala-Ala mutation would describe a mutation(s)from phenylalanine to alanine at position 234 and/or a mutation fromleucine to alanine at position 235. In another embodiment, the antibodyor Fc fusion protein comprises an IgG1 framework, wherein the Ala-Alamutation would describe a mutation(s) from leucine to alanine atposition 234 and/or a mutation from leucine to alanine at position 235.The antibody or Fc fusion protein may alternatively or additionallycarry other mutations, including the point mutation K322A in the CH2domain (Hezareh et al. 2001 J. Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may bemodified to either enhance or inhibit complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region (see,e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved or reduced internalizationcapability and/or increased or decreased complement-mediated cellkilling. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes,B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature322: 738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821;and WO94/29351.

6. Methods and Compositions for Modulating Angiogenesis and CertainDisorders

The disclosure provides methods of inhibiting angiogenesis in a mammalby administering to a subject an effective amount of a an ALK1 ECDpolypeptide, such as an ALK1-Fc fusion protein, a DAN protein, such as aDAN-Fc fusion protein, or an antibody disclosed herein, such as anantibody against GDF5, GDF6, GDF7, BMP9, BMP10, or the ECD of ALK1, ornucleic acid antagonists (e.g., antisense or siRNA) of any of theforegoing hereafter collectively referred to as “therapeutic agents”.The data presented indicate specifically that the anti-angiogenictherapeutic agents disclosed herein may be used to inhibit angiogenesisin the eye of a mammal. It is expected that these therapeutic agentswill also be useful in inhibiting angiogenesis in bones and joints, andin tumors, particularly tumors associated with bones and joints.

Angiogenesis associated diseases include, but are not limited to,angiogenesis-dependent cancer, including, for example, solid tumors,blood born tumors such as leukemias, and tumor metastases; benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; andangiofibroma.

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing a cancer (tumor), and particularlysuch cancers as are known to rely on angiogenic processes to supportgrowth. Unlike most anti-angiogenic agents, ALK1 ECD polypeptides affectangiogenesis that is stimulated by multiple factors. This is highlyrelevant in cancers, where a cancer will frequently acquire multiplefactors that support tumor angiogenesis. Thus, the therapeutic agentsdisclosed herein will be particularly effective in treating tumors thatare resistant to treatment with a drug that targets a single angiogenicfactor (e.g., bevacizumab, which targets VEGF). As demonstrated herein,an ALK1-Fc fusion protein is effective in reducing the pathologicaleffects of multiple myeloma. Multiple myeloma is widely recognized as acancer that includes a significant angiogenic component. Accordingly, itis expected that ALK1-Fc fusion proteins and other therapeutic agentsdisclosed herein will be useful in treating multiple myeloma and othertumors associated with the bone. As demonstrated herein, therapeuticagents disclosed herein may be used to ameliorate the bone damageassociated with multiple myeloma, and therefore may be used toameliorate bone damage associated with bone metastases of other tumors,such as breast or prostate tumors. As noted herein, the GDF5-7 ligandsare highly expressed in bone, and, while not wishing to be limited toany particular mechanism, interference with these ligands may disruptprocesses that are required for tumor development in bone.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject methods of the disclosure can beused alone. Alternatively, the subject methods may be used incombination with other conventional anti-cancer therapeutic approachesdirected to treatment or prevention of proliferative disorders (e.g.,tumor). For example, such methods can be used in prophylactic cancerprevention, prevention of cancer recurrence and metastases aftersurgery, and as an adjuvant of other conventional cancer therapy. Thepresent disclosure recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of a subjectpolypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent disclosed herein is administered incombination with another conventional anti-neoplastic agent, eitherconcomitantly or sequentially, such therapeutic agent may enhance thetherapeutic effect of the anti-neoplastic agent or overcome cellularresistance to such anti-neoplastic agent. This allows decrease of dosageof an anti-neoplastic agent, thereby reducing the undesirable sideeffects, or restores the effectiveness of an anti-neoplastic agent inresistant cells.

According to the present disclosure, the antiangiogenic agents describedherein may be used in combination with other compositions and proceduresfor the treatment of diseases. For example, a tumor may be treatedconventionally with surgery, radiation or chemotherapy combined with theALK1 or ALK1 ligand antagonist and then the antagonist may besubsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize any residual primary tumor.

Angiogenesis-inhibiting agents can also be given prophylactically toindividuals known to be at high risk for developing new or re-currentcancers. Accordingly, an aspect of the disclosure encompasses methodsfor prophylactic prevention of cancer in a subject, comprisingadministrating to the subject an effective amount of an ALK1 or ALK1ligand antagonist and/or a derivative thereof, or anotherangiogenesis-inhibiting agent of the present disclosure.

As demonstrated herein, ALK1-Fc is effective for diminishing thephenotype of a murine model of rheumatoid arthritis. Accordingly,therapeutic agents disclosed herein may be used for the treatment ofrheumatoid arthritis and other type of bone or joint inflammation.

Certain normal physiological processes are also associated withangiogenesis, for example, ovulation, menstruation, and placentation.The angiogenesis inhibiting proteins of the present disclosure areuseful in the treatment of disease of excessive or abnormal stimulationof endothelial cells. These diseases include, but are not limited to,intestinal adhesions, atherosclerosis, scleroderma, and hypertrophicscars, i.e., keloids. They are also useful in the treatment of diseasesthat have angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minalia quintosa) and ulcers (Helicobacter pylori).

General angiogenesis inhibiting proteins can be used as a birth controlagent by reducing or preventing uterine vascularization required forembryo implantation. Thus, the present disclosure provides an effectivebirth control method when an amount of the inhibitory protein sufficientto prevent embryo implantation is administered to a female. In oneaspect of the birth control method, an amount of the inhibiting proteinsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method. While notwanting to be bound by this statement, it is believed that inhibition ofvascularization of the uterine endometrium interferes with implantationof the blastocyst. Similar inhibition of vascularization of the mucosaof the uterine tube interferes with implantation of the blastocyst,preventing occurrence of a tubal pregnancy. Administration methods mayinclude, but are not limited to, pills, injections (intravenous,subcutaneous, intramuscular), suppositories, vaginal sponges, vaginaltampons, and intrauterine devices. It is also believed thatadministration of angiogenesis inhibiting agents of the presentdisclosure will interfere with normal enhanced vascularization of theplacenta, and also with the development of vessels within a successfullyimplanted blastocyst and developing embryo and fetus.

In the eye, angiogenesis is associated with, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, and retrolental fibroplasias. Thetherapeutic agents disclosed herein may be administered intra-ocularlyor by other local administration to the eye. Furthermore, as shown inthe Examples, ALK1-Fc may be administered systemically and yet have thedesired effect on ocular angiogenesis.

Other diseases associated with angiogenesis in the eye include, but arenot limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andcorneal graph rejection. sickle cell anemia, sarcoid, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infections,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, Eales disease, Bechets disease, infections causing aretinitis or choroiditis, presumed ocular histoplasmosis, Bests disease,myopia, optic pits, Stargarts disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovasculariation of the angle)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a therapeutic agent, or by insertionof a sustained release device that releases a therapeutic agent. Atherapeutic agent may be delivered in a pharmaceutically acceptableophthalmic vehicle, such that the compound is maintained in contact withthe ocular surface for a sufficient time period to allow the compound topenetrate the corneal and internal regions of the eye, as for examplethe anterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.The pharmaceutically-acceptable ophthalmic vehicle may, for example, bean ointment, vegetable oil or an encapsulating material. Alternatively,the therapeutic agents of the disclosure may be injected directly intothe vitreous and aqueous humour. In a further alternative, the compoundsmay be administered systemically, such as by intravenous infusion orinjection, for treatment of the eye.

One or more therapeutic agents can be administered. The methods of thedisclosure also include co-administration with other medicaments thatare used to treat conditions of the eye. When administering more thanone agent or a combination of agents and medicaments, administration canoccur simultaneously or sequentially in time. The therapeutic agentsand/or medicaments may be administered by different routes ofadministration or by the same route of administration. In oneembodiment, a therapeutic agent and a medicament are administeredtogether in an ophthalmic pharmaceutical formulation.

In one embodiment, a therapeutic agent is used to treat a diseaseassociated with angiogenesis in the eye by concurrent administrationwith other medicaments that act to block angiogenesis by pharmacologicalmechanisms. Medicaments that can be concurrently administered with atherapeutic agent of the disclosure include, but are not limited to,pegaptanib (Macugen™), ranibizumab (Lucentis™), squalamine lactate(Evizon™), heparinase, and glucocorticoids (e.g. Triamcinolone). In oneembodiment, a method is provided to treat a disease associated withangiogenesis is treated by administering an ophthalmic pharmaceuticalformulation containing at least one therapeutic agent disclosed hereinand at least one of the following medicaments: pegaptanib (Macugen™),ranibizumab (Lucentis™), squalamine lactate (Evizon™), heparinase, andglucocorticoids (e.g. Triamcinolone).

7. Formulations and Effective Doses

The therapeutic agents described herein may be formulated intopharmaceutical compositions. Pharmaceutical compositions for use inaccordance with the present disclosure may be formulated in conventionalmanner using one or more physiologically acceptable carriers orexcipients. Such formulations will generally be substantially pyrogenfree, in compliance with most regulatory requirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the ALK1 signalingantagonists which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., ALK1 ECD polypeptides or any of theantibodies disclosed herein) in the methods disclosed herein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more ALK1 ECD polypeptides or otherantibodies in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

In one embodiment, the antibodies and ALK1 ECD proteins disclosed hereinare administered in an ophthalmic pharmaceutical formulation. In someembodiments, the ophthalmic pharmaceutical formulation is a sterileaqueous solution, preferable of suitable concentration for injection, ora salve or ointment. Such salves or ointments typically comprise one ormore antibodies or ALK1 ECD proteins disclosed herein dissolved orsuspended in a sterile pharmaceutically acceptable salve or ointmentbase, such as a mineral oil-white petrolatum base. In salve or ointmentcompositions, anhydrous lanolin may also be included in the formulation.Thimerosal or chlorobutanol are also preferably added to such ointmentcompositions as antimicrobial agents. In one embodiment, the sterileaqueous solution is as described in U.S. Pat. No. 6,071,958.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range. Additional medicaments may be added to the formulation.These include, but are not limited to, pegaptanib, heparinase,ranibizumab, or glucocorticoids. The ophthalmic pharmaceuticalformulation according to the disclosure is prepared by asepticmanipulation, or sterilization is performed at a suitable stage ofpreparation.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

EXAMPLES Example 1 Expression of ALK1-Fc Fusion Proteins

Applicants constructed a soluble ALK1 fusion protein that has theextracellular domain of human ALK1 fused to a human Fc or mouse ALK1fused to a murine Fc domain with a minimal linker in between. Theconstructs are referred to as hALK1-Fc and mALK1-Fc, respectively.

hALK1-Fc is shown as purified from CHO cell lines in FIG. 3 (SEQ ID NO:3). Notably, while the conventional C-terminus of the extracellulardomain of human ALK1 protein is amino acid 118 of SEQ ID NO:1, we havedetermined that it is desirable to avoid having a domain that ends at aglutamine residue. Accordingly, the portion of SEQ ID NO:3 that derivesfrom human ALK1 incorporates two residues c-terminal to Q118, a leucineand an alanine. The disclosure therefore provides ALK1 ECD polypeptides(including Fc fusion proteins) having a c-terminus of the ALK1 derivedsequence that is anywhere from 1 to 5 amino acids upstream (113-117relative to SEQ ID NO:1) or downstream (119-123) of Q118.

The hALK1-Fc and mALK1-Fc proteins were expressed in CHO cell lines.Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 7) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 8)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 9)MTLGSPRKGLLMLLMALVTQG.

The selected form employs the TPA leader and has the unprocessed aminoacid sequence shown in FIG. 4 (SEQ ID NO:5).

This polypeptide is encoded by the nucleic acid sequence shown in FIG. 4(SEQ ID NO:4).

Purification can be achieved by a series of column chromatography steps,including, for example, three or more of the following, in any order:protein A chromatography, Q sepharose chromatography, phenylsepharosechromatography, size exclusion chromatography, and cation exchangechromatography. The purification can be completed with viral filtrationand buffer exchange. The hALK1-Fc protein was purified to a purityof >98% as determined by size exclusion chromatography and >95% asdetermined by SDS PAGE.

Example 2 Identification of ALK1-Fc Ligands

ALK1 is a type 1 receptors for members of the TGFβ family. A variety ofmembers of the TGFβ family were tested for binding to a human ALK1-Fcfusion protein, using a Biacore™ system. TGFβ itself, GDF5, GDF11, BMP2and BMP4 all failed to show substantial binding to the hALK1-Fc protein.BMP2 and BMP4 showed limited binding. GDF5, GDF7 and BMP9 showed bindingwith K_(D) values of approximately 5×10⁻⁸ M, 5×10⁻⁸ M and 1×10⁻¹⁰ M,respectively. Based on the similarity of GDF5 and GDF7 to GDF6, it isexpected that GDF6 will bind with similar affinity. BMP10 is closelyrelated to BMP9 and is also expected to bind with similar affinity.

Example 3 Characterization of ALK1-Fc and Anti-ALK1 Antibody Effects onEndothelial Cells

Using a luciferase reporter construct under the control of foursequential consensus SBE sites (SBE4-luc), which are responsive toSmad1/5/8-mediated signaling, we measured BMP-9 mediated activity in thepresence and absence of hALK1-Fc drug or neutralizing ALK1 specificmonoclonal antibody in HMVEC cells. HMVEC cells were stimulated withrhBMP-9 (50 ng/ml), which induced Smad1/5/8-mediated transcriptionalactivation, evidenced here by the increase in SBE4-luc modulatedtranscriptional upregulation. When added, the hALK1-Fc compound (10μg/ml) or antibody (10 μg/ml) diminished this transcriptional response,each by nearly 60%, indicating that the presence of ALK1-Fcsignificantly reduces BMP9 signaling, and moreover, that the BMP9signaling is related to ALK1 activity.

Activation of SMAD phosphorylation is commonly used to assay activationof upstream activin receptors. ALK1 is known to modulate phosphorylationof SMAD proteins 1, 5 and 8 upon activation by its ligand. Here, weadded rhBMP-9 (50 ng/ml) to initiate SMAD phosphorylation in HUVECcells, a human endothelial cell line which innately expresses ALK1receptor, over a timecourse of 30 minutes. Phosphorylation of SMAD 1/5/8was seen 5 minutes after treatment of cells with ligand andphosphorylation was maintained for the entirety of the 30 minute period.In the presence of relatively low concentrations of hALK1-Fc (250ng/ml), SMAD 1/5/8 phosphorylation was reduced, confirming that thisagent inhibits Smad1/5/8 activation in endothelial cells.

In order to evaluate the angiogenic effect of ALK1-Fc in an in vitrosystem, we assayed the effectiveness of the compound in reducing tubeformation of endothelial cells on a Matrigel substrate. This techniqueis commonly used to assess neovascularization, giving both rapid andhighly reproducible results. Endothelial Cell Growth Supplement (ECGS)is used to induce the formation of microvessels from endothelial cellson Matrigel, and the efficacy of anti-angiogenic compounds are thengauged as a reduction of cord formation in the presence of both the drugand ECGS over an 18 hour timecourse. As expected, addition of ECGS (200ng/ml) induced significant cord formation, as compared to the negativecontrol (no treatment added), which indicates basal levels ofendothelial cell cord formation produced on Matrigel substrate (FIG. 5).Upon addition of either hALK1-Fc (100 ng/ml) or mALK1-Fc (100 ng/ml),cord formation was visibly reduced. Final quantification of vessellength in all samples revealed that every concentration of hALK1-fc ormALK1-Fc reduced neovascularization to basal levels. Additionally,hALK1-Fc and mALK1-Fc in the presence of the strongly pro-angiogenicfactor ECGS maintained strong inhibition of neovascularizationdemonstrating even more potent anti-angiogenic activity than thenegative control endostatin (100 ng/ml).

Example 4 CAM Assays

VEGF and FGF are well-known to stimulate angiogenesis. A CAM (chickchorioallantoic membrane) assay system was used to assess the angiogeniceffects of GDF7. As shown in FIG. 6, GDF7 stimulates angiogenesis with apotency that is similar to that of VEGF. Similar results were observedwith GDF5 and GDF6.

ALK1-Fc fusions were tested for anti-angiogenic activity in the CAMassay. These fusion proteins showed a potent anti-angiogenic effect onangiogenesis stimulated by VEGF, FGF and GDF7. See FIG. 7. BMP9 and PDGFshowed a relatively poor capability to induce angiogenesis in thisassay, but such angiogenesic effect of these factors was nonethelessinhibited by ALK1.

ALK1-Fc proteins and a commercially available, anti-angiogenic anti-VEGFmonoclonal antibody were compared in the CAM assay. The ALK1-Fc proteinshad similar potency as compared to anti-VEGF. The anti-VEGF antibodybevacizumab is currently used in the treatment of cancer and maculardegeneration in humans. See FIG. 8.

Interestingly, an anti-ALK1 antibody (R&D Systems) failed tosignificantly inhibit angiogenesis in this assay system. We expect thatthis may reflect the difference in the ALK1 sequence in differentspecies.

Example 5 Mouse Corneal Micropocket Assay

The mouse corneal micropocket assay was used to assess the effects ofALK1-Fc on angiogenesis in the mouse eye. hALK1-Fc, administeredintraperitoneally, significantly inhibited ocular angiogenesis. As shownin FIG. 9, hALK1-Fc inhibited ocular angiogenesis to the same degree asanti-VEGF. hALK1-Fc and anti-VEGF were used at identical weight/weightdosages. Similar data were obtained when a Matrigel plug impregnatedwith VEGF was implanted in a non-ocular location.

These data demonstrate that high affinity ligands for ALK1 promoteangiogenesis and that an ALK1-Fc fusion protein has potentanti-angiogenic activity. The ligands for ALK1 fall into two categories,with the GDF5, 6, 7 grouping having an intermediate affinity for ALK1and the BMP9, 10 grouping having a high affinity for ALK1.

GDF5, 6 and 7 are primarily localized to bone and joints, while BMP9 iscirculated in the blood. Thus, there appears to be a pro-angiogenicsystem of the bones and joints that includes ALK1, GDF5, 6 and 7 and asystemic angiogenic system that includes ALK1 and BMP9 (and possiblyBMP10).

Example 6 Murine Model of Rheumatoid Arthritis

The murine collagen-induced arthritis model is a well-accepted model ofrheumatoid arthritis. In this study, groups of 10 mice were treated withvehicle, anti-VEGF (bevacizumab—as a negative control, becausebevacizumab does not inhibit murine VEGF), or doses of mALK1-Fc(“RAP-041”) at 1 mg/kg, 10 mg/kg or 25 mg/kg. Following the collagenboost on day 21 arthritic scores (see FIG. 10) and paw swelling steadilyincreased in all groups, peaking around day 38. Mice treated withmALK1-Fc (“RAP-041”) showed reduced scores for both characteristics,particularly at the highest dose (25 mg/kg), although the reduction didnot achieve statistical significance. Nonetheless, a dose-related trendis apparent.

By study termination at day 42 the incidence of arthritis had reached10/10 in the vehicle control treated mice, 9/10 in the bevacizumabtreated mice, 8/10 in the mALK1-Fc at 1 mg/kg treated group and 9/10 inthe mALK1-Fc 10 mg/kg treated group. In the mALK1-Fc 25 mg/kg treatedgroup disease incidence was lower at 6/10.

Example 7 Murine Model of Multiple Myeloma

Multiple myeloma is a cancer primarily of the bone that is associatedwith substantial bone loss. The 5T2MM model of myeloma in mice is basedon the use of tumor cells (5T2MM cells) from a type of spontaneous tumorthat develops in aged mice and causes effects in mice that are similarto those seen in human multiple myeloma patients. See, e.g.,Vanderkerken et al., Methods Mol. Med. 2005; 113:191-205. mALK1-Fc wastested for effects in this model.

5T2MM cells injected into C57BL/KaLwRij mice promotes an increase inosteoclast surface, the formation of osteolytic lesions and caused adecrease in bone area. Bone disease is associated with a decrease inosteoblast number, osteoblast surface and a reduction in mineralization.

Mice bearing 5T2MM cells were treated with mALK1-Fc (RAP-041) (10 mg/kg,i.p. twice weekly), or a vehicle, from the time of 5T2MM injection, fora total of 12 weeks. MicroCT analysis of the proximal tibia and lumbarvertebrae demonstrated a statistically significant reduction incancellous bone volume and trabecular number in 5T2MM-bearing micecompared to naïve mice (bone volume reduced by 40% relative to controls;trabecular number reduced by 40%). RAP-041 completely prevented5T2MM-induced decreases in bone volume and trabecular number whencompared to vehicle treated mice (treated mice had a bone volume of 120%relative to untumored controls and trabecular number of 115% relative tountumored controls). Additionally, the tumor treated mice developedlytic bone lesions that were detected by microCT. mALK1-Fc treatmentreduced the number of lytic bone lesions by 50% relative to vehicletreated mice.

Based on the anti-angiogenic effects of ALK1-Fc, we infer that theprotective effect for bone provided by this agent is a consequence ofdiminished tumor growth.

Therefore, ALK1-Fc may be used to treat multiple myeloma and to decreasethe effects of bone disease resulting from this tumor type.

Example 8 Ligand Binding Characteristics of DAN

DAN is a member of a family of secreted cystine knot proteins thatinhibit BMP activity. DAN is known to bind to and antagonize GDF5. Wedetermined that DAN also binds tightly to GDF7, but not to BMP9. Thus,we conclude that DAN inhibits the suite of bone and joint localizedligands for ALK1, and DAN is expected to be a potent antagonist of boneand joint related angiogenesis. Thus DAN may be useful in treatingcancers of the bone, e.g., multiple myeloma and bone metastases, as wellas rheumatoid arthritis and osteoarthritis.

Taken together, the findings disclosed in these Examples providenumerous reagents, described herein, for inhibiting angiogenesis invivo, and particularly ocular angiogenesis. These findings also indicatethat agents targeted to GDF5, 6 and 7 can be used to selectively inhibitbone and joint angiogenesis. These findings further indicate that suchagents can be used to treat cancers and rheumatoid arthritis.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject inventions are explicitlydisclosed herein, the above specification is illustrative and notrestrictive. Many variations of the inventions will become apparent tothose skilled in the art upon review of this specification and theclaims below. The full scope of the inventions should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

We claim:
 1. An ALK1-Fc fusion protein comprising: a polypeptide havingan amino acid sequence that is at least 97% identical to the sequence ofamino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fcportion of an immunoglobulin, and wherein the ALK1-Fc fusion proteinbinds to GDF5, GDF7 and BMP9 with a K_(D) of less than 1×10⁻⁷ M andbinds to TGFβ-1 with a K_(D) of greater than 1×10⁻⁶.
 2. The ALK1-Fcfusion protein, wherein the Fc portion is an Fc portion of a humanIgG
 1. 3. An ALK1-Fc fusion protein comprising the amino acid sequenceof SEQ ID NO:
 3. 4. An ALK1-Fc fusion protein that is produced byexpression of the nucleic acid of SEQ ID NO:4 in a mammalian cell line.5. The ALK1-Fc fusion protein of claim 4, wherein the mammalian cellline is a Chinese Hamster Ovary (CHO) cell line.
 6. A pharmaceuticalpreparation that is substantially pyrogen free, comprising an ALK1-Fcfusion protein of claim
 5. 7. An antibody that binds to an ALK1polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibitsthe binding of at least one ALK1 ligand selected from the groupconsisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.
 8. The antibody ofclaim 7, wherein the antibody binds to the ALK1 polypeptide with a K_(D)of less than 5×10⁻⁸ M.
 9. The antibody of claim 7, wherein the antibodybinds to the ALK1 polypeptide with a K_(D) of less than 1×10⁻¹⁰ M. 10.The antibody of claim 8, wherein the antibody inhibits angiogenesisstimulated by at least one ALK1 ligand selected from the groupconsisting of: GDF5, GDF6 and GDF7.
 11. The antibody of claim 10,wherein the antibody inhibits binding of BMP9 and BMP10 to ALK1.
 12. Apharmaceutical preparation that is substantially pyrogen free comprisingthe antibody of claim
 7. 13. A method of inhibiting angiogenesis in amammal, the method comprising, administering to the mammal an effectiveamount of an ALK1 ECD protein.
 14. The method of claim 13, wherein theALK-1 ECD protein is an ALK1-Fc fusion protein.
 15. The method of claim14, wherein the ALK1-Fc fusion protein comprises a polypeptide having anamino acid sequence that is at least 90% identical to the sequence ofamino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fcportion of an immunoglobulin, and wherein the ALK1-Fc fusion proteinbinds to TGFβ-1 with a K_(D) of greater than 1×10⁻⁶.
 16. The method ofclaim 13, wherein the ALK1 ECD protein binds to one or more ALK1 ligandsselected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.17. The method of claim 13, wherein the ALK1 ECD polypeptide comprisesan amino acid sequence that is at least 90% identical to the sequence ofamino acids corresponding to amino acids 34-95 of SEQ ID NO:1.
 18. Themethod of claim 13, wherein the ALK1 ECD comprises an amino acidsequence encoded by a nucleic acid that hybridized under stringenthybridization conditions to nucleotides 100-285 of SEQ ID NO:2 or avariant of nucleotides 100-285 of SEQ ID NO:2 that has the same codingsequence.
 19. The method of claim 14, wherein the ALK1-Fc fusion proteinhas a sequence of SEQ ID NO:3.
 20. The method of claim 13, wherein theALK1 ECD fusion protein is delivered intravenously or locally to theeye.
 21. The method of claim 13, wherein the method further comprisesadministering a second agent that inhibits angiogenesis.
 22. The methodof claim 13, wherein the angiogenesis to be inhibited is angiogenesisoccurring: in the eye of the mammal, in a tumor or in a bone or joint.23. A method of inhibiting angiogenesis in a mammal, the methodcomprising, administering to the mammal an effective amount of anantibody that binds to an ALK1 polypeptide consisting of amino acids22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALK1ligand selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 andBMP10.
 24. The method of claim 23, wherein the antibody binds to theALK1 polypeptide with a K_(D) of less than 5×10⁻⁸ M.
 25. The method ofclaim 23, wherein the antibody binds to the ALK1 polypeptide with aK_(D) of less than 1×10⁻¹⁰ M.
 26. The method of claim 23, wherein theantibody inhibits angiogenesis stimulated by at least one ALK1 ligandselected from the group consisting of: GDF5, GDF6 and GDF7.
 27. Themethod of claim 23, wherein the antibody inhibits binding of ALK1 to anALK1 ligand, wherein the ALK1 ligand is selected from the groupconsisting of: BMP9 and BMP10.
 28. The method of claim 23, wherein theantibody is delivered intravenously.
 29. The method of claim 23, whereinthe method further comprises administering a second agent that inhibitsangiogenesis.
 30. The method of claim 23, wherein the angiogenesis to beinhibited is angiogenesis occurring: in the eye of the mammal, in atumor or in a bone or joint.
 31. An antibody that binds to an ALK1ligand and inhibits the binding of the ALK1 ligand to ALK1, wherein theALK1 ligand is selected from the group consisting of GDF5, GDF6 andGDF7.
 32. The antibody of claim 31, wherein the antibody binds to theALK1 ligand with a K_(D) of less than 5×10⁻⁸ M.
 33. The antibody ofclaim 31, wherein the antibody inhibits angiogenesis stimulated by theALK1 ligand.
 34. A pharmaceutical preparation comprising the antibody ofclaim
 31. 35. An antibody that binds to an ALK1 ligand and inhibits thebinding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is selectedfrom the group consisting of BMP9 and BMP10.
 36. The antibody of claim35, wherein the antibody binds to the ALK1 ligand with a K_(D) of lessthan 1×10⁻¹⁰ M.
 37. A pharmaceutical preparation comprising the antibodyof claim
 35. 38. A method of inhibiting angiogenesis in a mammal, themethod comprising, administering to the mammal an effective amount of anantibody that binds to an ALK1 ligand and inhibits the binding of theALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the groupconsisting of GDF5, GDF6, GDF7, BMP9 and BMP10.
 39. The method of claim38, wherein the antibody inhibits angiogenesis stimulated by at leastone ALK1 ligand selected from the group consisting of: GDF5, GDF6 andGDF7.
 40. The method of claim 38, wherein the antibody inhibitsangiogenesis stimulated by at least one ALK1 ligand selected from thegroup consisting of: BMP9 and BMP10.
 41. The method claim 38, whereinthe antibody is delivered intravenously.
 42. The method of claim 38,wherein the method further comprises administering a second agent thatinhibits angiogenesis.
 43. The method of claim 38, wherein theangiogenesis to be inhibited is angiogenesis occurring: in the eye ofthe mammal, in a tumor or in a bone or joint.
 44. Use of an ALK1 ECDpolypeptide or nucleic acids encoding the same in the manufacture of amedicament for the inhibition of angiogenesis in a mammal.
 45. The useof claim 44, wherein the ALK1 ECD polypeptide is an ALK-1 Fc fusionprotein.
 46. The use of claim 45, wherein the ALK-1 Fc fusion proteincomprises a polypeptide having an amino acid sequence that is at least90% identical to the sequence of amino acids 22-118 of SEQ ID NO:1,which polypeptide is fused to an Fc portion of an immunoglobulin, andwherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K_(D) ofgreater than 1×10⁻⁶.
 47. Use of an antibody that binds to ALK1 ornucleic acids encoding the same in the manufacture of a medicament forthe inhibition of angiogenesis in a mammal, wherein the antibody bindsto an ALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1and inhibits the binding of at least one ALK1 ligand selected from thegroup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.
 48. Use of anantibody that binds to an ALK1 ligand and inhibits the binding of theALK1 ligand to ALK1, or nucleic acids encoding the same, in themanufacture of a medicament for the inhibition of angiogenesis in amammal, wherein the antibody binds to an ALK1 ligand selected from thegroup consisting of GDF5, GDF6, GDF7, BMP9 and BMP10.
 49. A method ofinhibiting angiogenesis in a mammal, the method comprising,administering to the mammal an effective amount of a DAN protein. 50.The method of claim 49, wherein the DAN protein is a DAN-Fc fusionprotein.
 51. The method of claim 49, wherein the DAN-Fc fusion proteincomprises a polypeptide having an amino acid sequence that is at least90% identical to the sequence of amino acids 21-125 of SEQ ID NO:10,which polypeptide is fused to an Fc portion of an immunoglobulin, andwherein the ALK1-Fc fusion protein binds to TGFβ-1 with a K_(D) ofgreater than 1×10⁻⁶.
 52. The method of claim 49, wherein the DAN proteinbinds to one or more ALK1 ligands selected from the group consisting of:GDF5, GDF6 and GDF7.
 53. The method of claim 49, wherein the DAN proteincomprises an amino acid sequence that is at least 90% identical to thesequence of amino acids corresponding to amino acids 17-180 of SEQ IDNO:10.
 54. The method of claim 49, wherein the DAN protein comprises anamino acid sequence encoded by a nucleic acid that hybridizes understringent hybridization conditions to nucleotides 153-467 of SEQ IDNO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has thesame coding sequence.
 55. The method of claim 49, wherein the methodfurther comprises administering a second agent that inhibitsangiogenesis.
 56. The method of claim 49, wherein the angiogenesis to beinhibited is angiogenesis occurring: in the eye of the mammal, in atumor or in a bone or joint.
 57. A method for treating rheumatoidarthritis in a mammal, the method comprising, administering to a mammalthat has rheumatoid arthritis an effective amount of an agent selectedfrom the group consisting of: (a) an ALK1 ECD protein; (b) an antibodythat binds to an ALK1 ligand and inhibits the binding of the ALK1 ligandto ALK1, wherein the ALK1 ligand is selected from the group consistingof GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to anALK1 polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 andinhibits the binding of at least one ALK1 ligand selected from the groupconsisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and (d) a DANpolypeptide.
 58. The method of claim 57, wherein the ALK-1 ECD proteinis an ALK1-Fc fusion protein.
 59. The method of claim 57, wherein theALK1-Fc fusion protein comprises a polypeptide having an amino acidsequence that is at least 90% identical to the sequence of amino acids22-118 of SEQ ID NO:1, which polypeptide is fused to an Fc portion of animmunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1with a K_(D) of greater than 1×10⁻⁶.
 60. The method of claim 57, whereinthe ALK1 ECD protein binds to one or more ALK1 ligands selected from thegroup consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.
 61. The method ofclaim 58, wherein the ALK1-Fc fusion protein has a sequence of SEQ IDNO:3.
 62. The method of claim 57, wherein the ALK1 ECD polypeptidecomprises an amino acid sequence that is at least 90% identical to thesequence of amino acids corresponding to amino acids 34-95 of SEQ IDNO:1.
 63. The method of claim 57, wherein the ALK1 ECD comprises anamino acid sequence encoded by a nucleic acid that hybridized understringent hybridization conditions to nucleotides 100-285 of SEQ ID NO:2or a variant of nucleotides 100-285 of SEQ ID NO:2 that has the samecoding sequence.
 64. The method of claim 57, wherein the antibody of (b)binds to the ALK1 polypeptide with a K_(D) of less than 5×10⁻⁸ M. 65.The method of claim 57, wherein the antibody of (b) binds to the ALK1polypeptide with a K_(D) of less than 1×10⁻¹⁰ M.
 66. The method of claim57, wherein the antibody of (b) inhibits angiogenesis stimulated by atleast one ALK1 ligand selected from the group consisting of: GDF5, GDF6and GDF7.
 67. The method of claim 57, wherein the antibody of (b)inhibits binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand isselected from the group consisting of: BMP9 and BMP10.
 68. The method ofclaim 57, wherein the antibody of (c) inhibits angiogenesis stimulatedby at least one ALK1 ligand selected from the group consisting of: GDF5,GDF6 and GDF7.
 69. The method of claim 57, wherein the antibody of (c)inhibits angiogenesis stimulated by at least one ALK1 ligand selectedfrom the group consisting of: BMP9 and BMP10.
 70. The method of claim57, wherein the DAN protein is a DAN-Fc fusion protein.
 71. The methodof claim 70, wherein the DAN-Fc fusion protein comprises a polypeptidehaving an amino acid sequence that is at least 90% identical to thesequence of amino acids 21-125 of SEQ ID NO:10, which polypeptide isfused to an Fc portion of an immunoglobulin, and wherein the ALK1-Fcfusion protein binds to TGFβ-1 with a K_(D) of greater than 1×10⁻⁶. 72.The method of claim 57, wherein the DAN protein binds to one or moreALK1 ligands selected from the group consisting of: GDF5, GDF6 and GDF7.73. The method of claim 57, wherein the DAN protein comprises an aminoacid sequence that is at least 90% identical to the sequence of aminoacids corresponding to amino acids 17-180 of SEQ ID NO:10.
 74. Themethod of claim 57, wherein the DAN protein comprises an amino acidsequence encoded by a nucleic acid that hybridizes under stringenthybridization conditions to nucleotides 153-467 of SEQ ID NO:11 or avariant of nucleotides 153-467 of SEQ ID NO:11 that has the same codingsequence.
 75. The method of claim 57, wherein the agent is deliveredintravenously.
 76. The method of claim 57, wherein the method furthercomprises administering a second agent that inhibits angiogenesis.
 77. Amethod for treating a tumor in a mammal, the method comprising,administering to a mammal that has a tumor an effective amount of anagent selected from the group consisting of: (a) an ALK1 ECD protein;(b) an antibody that binds to an ALK1 ligand and inhibits the binding ofthe ALK1 ligand to ALK1, wherein the ALK1 ligand is selected from thegroup consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibodythat binds to an ALK1 polypeptide consisting of amino acids 22-118 ofSEQ ID NO:1 and inhibits the binding of at least one ALK1 ligandselected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10;and (d) a DAN polypeptide.
 78. The method of claim 77, wherein the ALK-1ECD protein is an ALK1-Fc fusion protein.
 79. The method of claim 78,wherein the ALK1-Fc fusion protein comprises a polypeptide having anamino acid sequence that is at least 90% identical to the sequence ofamino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to an Fcportion of an immunoglobulin, and wherein the ALK1-Fc fusion proteinbinds to TGFβ-1 with a K_(D) of greater than 1×10⁻⁶.
 80. The method ofclaim 77, wherein the ALK1 ECD protein binds to one or more ALK1 ligandsselected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10.81. The method of claim 78, wherein the ALK1-Fc fusion protein has asequence of SEQ ID NO:3.
 82. The method of claim 77, wherein the ALK1ECD polypeptide comprises an amino acid sequence that is at least 90%identical to the sequence of amino acids corresponding to amino acids34-95 of SEQ ID NO:1.
 83. The method of claim 77, wherein the ALK1 ECDcomprises an amino acid sequence encoded by a nucleic acid thathybridized under stringent hybridization conditions to nucleotides100-285 of SEQ ID NO:2 or a variant of nucleotides 100-285 of SEQ IDNO:2 that has the same coding sequence.
 84. The method of claim 77,wherein the antibody of (b) binds to the ALK1 polypeptide with a K_(D)of less than 5×10⁻⁸ M.
 85. The method of claim 77, wherein the antibodyof (b) binds to the ALK1 polypeptide with a K_(D) of less than 1×10⁻¹⁰M.
 86. The method of claim 77, wherein the antibody of (b) inhibitsangiogenesis stimulated by at least one ALK1 ligand selected from thegroup consisting of: GDF5, GDF6 and GDF7.
 87. The method of claim 77,wherein the antibody of (b) inhibits binding of ALK1 to an ALK1 ligand,wherein the ALK1 ligand is selected from the group consisting of: BMP9and BMP10.
 88. The method of claim 77, wherein the antibody of (c)inhibits angiogenesis stimulated by at least one ALK1 ligand selectedfrom the group consisting of: GDF5, GDF6 and GDF7.
 89. The method ofclaim 77, wherein the antibody of (c) inhibits angiogenesis stimulatedby at least one ALK1 ligand selected from the group consisting of: BMP9and BMP10.
 90. The method of claim 77, wherein the DAN protein is aDAN-Fc fusion protein.
 91. The method of claim 90, wherein the DAN-Fcfusion protein comprises a polypeptide having an amino acid sequencethat is at least 90% identical to the sequence of amino acids 21-125 ofSEQ ID NO:10, which polypeptide is fused to an Fc portion of animmunoglobulin, and wherein the ALK1-Fc fusion protein binds to TGFβ-1with a K_(D) of greater than 1×10⁻⁶.
 92. The method of claim 77, whereinthe DAN protein binds to one or more ALK1 ligands selected from thegroup consisting of: GDF5, GDF6 and GDF7.
 93. The method of claim 77,wherein the DAN protein comprises an amino acid sequence that is atleast 90% identical to the sequence of amino acids corresponding toamino acids 17-180 of SEQ ID NO:10.
 94. The method of claim 77, whereinthe DAN protein comprises an amino acid sequence encoded by a nucleicacid that hybridizes under stringent hybridization conditions tonucleotides 153-467 of SEQ ID NO:11 or a variant of nucleotides 153-467of SEQ ID NO:11 that has the same coding sequence.
 95. The method ofclaim 77, wherein the agent is delivered intravenously.
 96. The methodof claim 77, wherein the method further comprises administering a secondagent that inhibits angiogenesis.
 97. The method of claim 77, whereinthe method further comprises administering a second agent that inhibitsangiogenesis.
 98. The method of claim 77, wherein the tumor isassociated with bone.
 99. The method of claim 77, wherein the tumor is amyeloma or a tumor that has metastasized to the bone.
 100. The method ofclaim 77, wherein the tumor is resistant to anti-VEGF therapy.
 101. Anophthalmic pharmaceutical formulation comprising an agent selected fromthe group consisting of: (a) an ALK1 ECD protein; (b) an antibody thatbinds to an ALK1 ligand and inhibits the binding of the ALK1 ligand toALK1, wherein the ALK1 ligand is selected from the group consisting ofGDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody that binds to an ALK1polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibitsthe binding of at least one ALK1 ligand selected from the groupconsisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and (d) a DANpolypeptide.
 102. A method of treating an angiogenesis related diseaseof the eye comprising administering systemically or to said eye apharmaceutical formulation comprising: an effective amount of an agentselected from the group consisting of: (a) an ALK1 ECD protein; (b) anantibody that binds to an ALK1 ligand and inhibits the binding of theALK1 ligand to ALK1, wherein the ALK1 ligand is selected from the groupconsisting of GDF5, GDF6, GDF7, BMP9 and BMP10; (c) an antibody thatbinds to an ALK1 polypeptide consisting of amino acids 22-118 of SEQ IDNO:1 and inhibits the binding of at least one ALK1 ligand selected fromthe group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10; and (d) a DANpolypeptide.